Epitope testing using soluble HLA

Abstract

The present invention relates generally to a methodology for assaying the binding of a peptide to an individual, specific, soluble HLA molecule using fluorescence polarization. The peptides utilized in the method may be identified by indirect methods utilizing T lymphocytes, or by a direct method of epitope discovery described herein.

What is claimed is: 
     
       1. A method of assaying the affinity of a peptide of interest for binding to an individual MHC class I molecule, the method comprising the steps of:
 labeling a first peptide with a fluorescent label, wherein the first peptide is known to bind to a specific MHC class I molecule; 
 activating individual soluble, specific MHC class I molecules by heating the individual soluble, specific MHC class I molecules for about 15 minutes at about 53° C. to cause the individual soluble, specific MHC class I molecules to release any endogenous peptide loaded therein, thereby activating the individual soluble, specific MHC class I molecules; 
 mixing the labeled first peptide and the activated individual soluble, specific MHC class I molecules together with different concentrations of a second peptide, wherein the second peptide is a peptide of interest, to provide individual soluble, specific MHC class I molecule-labeled first peptide complexes and individual soluble, specific MHC class I molecule-second peptide complexes, wherein the individual soluble, specific MHC class I molecule-first peptide complexes are detectable by fluorescence polarization; and 
 determining a binding affinity of the peptide of interest for the individual soluble, specific MHC class I molecule by determining an inhibitory concentration of the second peptide that inhibits 50% binding of the first peptide to the individual soluble, specific MHC class I molecule. 
 
     
     
       2. The method of  claim 1  wherein, in the method of providing individual soluble, specific MHC class I molecules, the individual soluble, specific MHC class I molecules are produced by the method comprising the steps of:
 obtaining genomic DNA or cDNA encoding at least one MHC class I molecule; 
 identifying an allele encoding an individual MHC class I molecule in the genomic DNA or cDNA; 
 PCR amplifying the allele encoding the individual MHC class I molecule in a locus specific manner such that a PCR product produced therefrom encodes a truncated, soluble form of the individual MHC class I molecule; 
 cloning the PCR product into an expression vector, thereby forming a construct that encodes the individual soluble, specific MHC class I molecule; 
 transfecting the construct into a cell line to provide a cell line containing a construct that encodes an individual soluble, specific MHC class I molecule, the cell line being able to naturally process proteins into peptide ligands capable of being loaded into antigen binding grooves of MHC class I molecules; 
 culturing the cell line under conditions which allow for expression of the individual soluble, specific MHC class I molecules from the construct, such conditions also allowing for endogenous loading of a peptide ligand into the antigen binding groove of each individual soluble, specific MHC class I molecule prior to secretion of the individual soluble, specific MHC class I molecules from the cell; and 
 isolating the secreted individual soluble, specific MHC class I molecules. 
 
     
     
       3. The method of  claim 2  wherein the construct further encodes a tag which is attached to the individual soluble, specific MHC class I molecule and aids in isolating the individual soluble, specific MHC class I molecule. 
     
     
       4. The method of  claim 3  wherein the tag is selected from the group consisting of a HIS tail and a FLAG tail. 
     
     
       5. A method of assaying the affinity of a peptide of interest for binding to an individual HLA class I molecule, the method comprising the steps of:
 (a) activating a pool of isolated, functionally active, recombinantly produced, secreted, individual soluble HLA class I trimolecular complexes for peptide exchange by application of thermal energy to thermodynamically destabilize the trimolecular complexes and thus allow the trimolecular complexes to participate in peptide exchange, each secreted, recombinant HLA class I trimolecular complex produced in a human cell line and comprising a recombinantly produced, soluble HLA class I heavy chain, non-covalently associated beta-2-microglobulin, and peptide, wherein the peptide is naturally produced by the human cell line and endogenously loaded into the trimolecular complex by the human cell line, and wherein each complex present in the pool comprises the same truncated, soluble HLA class I heavy chain, and wherein the activation of the trimolecular complexes thermodynamically destabilizes the complexes and thus allows the complexes to participate in peptide exchange; 
 (b) mixing together:
 (i) the pool of activated, secreted, recombinant HLA class I trimolecular complexes of (a); 
 (ii) a labeled first peptide known to bind to the specific HLA class I molecule of the pool of HLA class I trimolecular complexes; and 
 (iii) a second peptide, wherein the second peptide is a peptide of interest; and 
 wherein the mixing of (i)-(iii) produces at least one of HLA class I trimolecular complexes containing the labeled first peptide and HLA class I trimolecular complexes containing the second peptide; and 
 
 (c) determining a binding affinity of the second peptide for the specific HLA class I molecule by determining an inhibitory concentration of the second peptide that inhibits 50% binding of the labeled first peptide to the activated soluble HLA class I trimolecular complex. 
 
     
     
       6. The method of  claim 5 , wherein the step of activating a pool of isolated, functionally active, recombinantly produced, secreted, individual soluble HLA class I trimolecular complexes for peptide exchange by application of thermal energy is further defined as heating the pool of individual soluble, HLA class I trimolecular complexes for about 15 minutes at about 53° C. to cause the pool of individual soluble, HLA class I trimolecular complexes to release any endogenous peptide loaded therein, thereby activating the individual soluble, HLA class I trimolecular complexes. 
     
     
       7. The method of  claim 5 , wherein the first peptide is labeled with a radiolabel or a fluorescent label. 
     
     
       8. The method of  claim 7 , wherein the first peptide is labeled with a fluorescent label, and the HLA class I trimolecular complexes containing the labeled first peptide are identified by fluorescence polarization. 
     
     
       9. The method of  claim 8 , wherein the fluorescent label is fluorescein isothiocyanate (FITC). 
     
     
       10. The method of  claim 8 , wherein (i)-(iii) are mixed together in the presence of excess beta-2-microglobulin. 
     
     
       11. The method of  claim 10 , wherein the ratio of added beta-2-microglobulin to recombinant soluble HLA class I heavy chain is about 3:1. 
     
     
       12. The method of  claim 10 , wherein (i)-(iii) are mixed together in the presence of bovine gamma globulin.